An ideal vaccine for the prevention of cholera is not yet available. Previous work in this project has resulted in the development of an attenuated live oral cholera vaccine, V. cholerae CVD 103-HgR. This vaccine confers strong protective immunity against experimental challenge with virulent V. cholerae O1 after a single dose. Although this vaccine is highly protective in North American volunteers and has been licensed in several highly developed countries for protection of travelers to cholera endemic countries, a recent field trial of this vaccine in Indonesia failed to show efficacy. The development of attenuated cholera vaccines has been plagued by the fact that V. cholerae strains deleted of the ctx genes encoding cholera toxin can still produce varying amounts of diarrhea and non-diarrheal symptoms such as headache, fever, abdominal cramps, and malaise in many individuals. Such symptoms are not seen with CVD 103-HgR, in all probability because this strain colonizes the human intestine at greatly reduced levels compared to the reactogenic, avidly colonizing ctx-negative strains. Although the reduced colonization of CVD 103-HgR was still sufficient to engender a protective immune response in North American volunteers whose small bowel intestinal flora is relatively sparse, it was not sufficient to induce a protective immune response in a cholera-endemic population with a heavy burden of small bowel intestinal flora which would compete against a live oral vaccine strain. The ability to construct a better-colonizing strain is hampered by the uncertainty as to what bacterial factor is responsible for the reactogenicity. Thus, the next period of support for this project will focus on characterizing the response of epithelial cells to adherent V. cholerae, establishing the role in reactogenicity of various cytolysins, proteases, other degradative enzymes, and other potential toxins revealed by the recently completed genome sequence of V. cholerae, and determining the V. cholerae genes that are specifically expressed during the course of human infection. These studies will use the broadest possible range of models to study host-pathogen interactions, including intestinal epithelial cell lines, freshly harvested human intestinal tissue, animal models, and human volunteer studies.